(Updated Nov 2007)
Background and History: I am an evolutionary biologist with a primary interest in molecular systematics and phylogeography. My research spans a diversity of biological systems and topics, conceptually unified by a phylogenetic perspective based on gene genealogies. Most of my work has been empirical, based on comparative DNA sequence and microsatellite data analysis, and focused on the evolution of fishes.
I started my lab at UNL in 1997, intending to develop an integrative phylogenetic research program, to document patterns and understand evolutionary processes spanning the range between recently diverging populations to large-scale macroevolutionary events in “deep time.” A CAREER grant from NSF awarded in 2000 allowed me to expand significantly my early studies on the phylogeny of characiform fishes to target a most ambitious goal, the phylogeny of “all fishes.” This topic has subsequently become an important part of my efforts while several other projects, not directly related to fish systematics, were unfolding in my lab (see Section III). Since 2005, I am the lead investigator of an international research coordination network to foster fish phylogenetics (DeepFin), funded by NSF’s RCN program. Networking activities led, among other things, to the assemblage of a multi-institutional research team that successfully attracted funding from NSF’s Assembling the Tree of Life Program in 2007. This team is now ready to conduct a large research effort and to lead the fish systematics community, for the first time, in a concerted effort to reconstruct the evolutionary origins of all living fishes. This project will dominate my research agenda for at least the next 5 years.
Rationale for a phylogenetic perspective: All forms of life share a common history due to the transmission of hereditary material over eons, generation after generation, and this history may be traced back in time through analysis of genetic lineages. The shape and distribution of gene genealogies in time and space are manifestations of current and historical processes that operate at diverse levels in the hierarchy of life. If properly interpreted, molecular genetic data may be used to infer organismal phylogenies (the tree of life) to study biological processes as ancient as the origin and diversification of the main forms of life and as recent as a familiar pedigree—as implied by the figure (from Avise et al. 1987, Annu. Rev. Ecol. Syst. 18).
My research has used this phylogenetic “lens” to address macro- and microevolutionary questions in a diversity of organisms, from viruses to vertebrate animals. In addition to the main focus on fish phylogenies, significant research directions in my lab also involved primates, host-parasite interactions, and HIV viruses. At the base of all these studies lies an extended conceptual framework that emphasizes the explanation of biological diversity in the context of a phylogenetic tree—“tree thinking.”
Funding and Students: Generous and ongoing funding from the National Science Foundation and other federal and state agencies has provided uninterrupted support for my research program since 1998. Overall, my individual and collaborative efforts have attracted $3.5 million to fund research activities at UNL. I owe much of this success to my collaborators and graduate students. To date, 5 PhD and 5 MS students have graduated from my lab under my direct supervision; currently, I am the major advisor for 3 more graduate students in good standing (2 PhD and 1 MS). Another 5 PhD students graduated from UNL with a significant component of their research performed in my lab under my supervision (serving as a co-advisor in many cases). Overall, I have served in more than 25 graduate student advisory committees at UNL. I also supervised 5 postdoctoral associates working on diverse projects. Finally, over the years, at least 15 undergraduate students from UNL acquired research experience in my lab, some of them co-authoring papers and then moving on to attend graduate school. Overall, we have published 48 peer-reviewed articles.
Two main lines of current research and miscellaneous projects: In addition to the main goal of establishing the phylogeny of all fishes (Section I), two secondary research programs are ongoing in my lab (Section II). These secondary projects focus on the origin of regional faunas, especially assemblages of fish communities, and how these communities have been shaped by biotic interactions and the effect of landscape formation. The approach we take is comparative phylogeography. One area of interest is Patagonia with its relatively modest freshwater ichthyofauna. The other one is Amazonia, with its mega diverse fish communities. These two projects have their roots in my long-standing interest to study phylogeography, the differentiation and distribution of genetic lineages in geographic space. By developing comparative phylogeographic perspectives for several species co-distributed across vast geographic areas, these projects intend to synthesize and test the effect of key historical events with major influence in the diversification and, ultimately, the composition of these biogeographic regions.
A few additional lines of research that we developed over the years are mentioned in Section III. My participation in these projects has been completed but they are worth mentioning here to illustrate the breadth of my research program, and also how a phylogenetic perspective can be used to illuminate a diversity of biological issues. I present briefly the main findings of studies on vertical transmission and evolution of HIV-1 in infants (in collaboration with my colleagues at the Nebraska Center for Virology) and of two doctoral theses of former students (genetic chimerism in marmosets and conservation genetics of catostomid fishes).
This project has been funded by the National Science Foundation since 2000, through a CAREER grant (DEB-9985045, from 2000 to 2006), a Research Coordination Network grant (DEB-0443470, from 2005-2010), and a Tree of Life grant (DEB-0732838, from 2007-2012). The goal of this research is to establish phylogenetic relationships among the main groups of all living ray-finned fishes (Actinopterygii).
Rationale:Of all living “fish” species, around 80 are jawless (“agnathans” such as hagfishes and lampreys), 850 species are cartilaginous (sharks, skates, rays, and chimeras), and the approximately 27,000 remaining are bony “fishes.” Excluding tetrapods and the few extant species of lungfishes and coelacanths, all living bony “fishes” are ray-finned fishes (Class Actinopterygii). The huge diversity of actinopterygians has been classified into 453 families in 44 orders (following Nelson 2006). The oldest actinopterygian fossils date back to the Silurian, more than 400 Million years ago. Some 20 supraordinal assemblages of varying degrees of inclusiveness have been proposed but most of them never have been properly tested. Currently, mostly loosely-defined “phylogenetic syntheses” are used to summarize hypotheses of relationships among the major groups of fishes. Popular views of fish phylogeny, compiled intuitively from many disconnected studies, appear in textbooks and catalogs (e.g. several editions of J.S. Nelson’s “Fishes of the World”); however, no comprehensive data set exists that could be used to test the branching pattern of the fish tree with explicit phylogenetic analyses. Homology assessment and identification of informative character sets that could yield phylogenetic information across the hierarchy of the fish tree remain challenging issues for morphologists. Molecular characters appear as a promising alternative but a set of “universal” molecular markers for fish phylogenetics still is under development. The long-term goal of this research is to compile a comprehensive comparative data set of morphological and molecular characters for a rich sample of representative taxa of actinopterygian fishes to construct well-supported phylogenetic hypotheses.
Background and progress to date: For more than 15 years, I have been constructing molecular phylogenies of fishes to examine evolutionary questions at different levels of the taxonomic spectrum, ranging from higher taxa, to species, to individuals within populations. At high-taxonomic level, my early work on the biogeography of characiform fishes (e.g., tetras, piranhas, hatchetfishes, and many other strictly freshwater groups) involved resolving relationships among families in the order Characiformes with more than 1500 species, currently distributed in two continents. These families are known to have diverged more than 100 million years ago, before the break-up of the Gondwanan supercontinent (Ortí 1997; Ortí and Meyer 1996, 1997). Molecular phylogenies at this level have provided a framework for testing biogeographic scenarios and for inferring diversification and extinction rates. During 1998 and 1999, molecular systematic work at high taxonomic level among cichlid fishes has been conducted in my lab at UNL by Izeni Farias, an exchange doctoral student from Brazil. Her work focused on the Neotropical radiation of cichlids but also studied relationships among cichlids distributed in Africa, Madagascar and India (Farias et al. 1999, 2000, 2001). Another graduate student continued this research on the heroin cichlid fishes that colonized Central and North America (Concheiro Pérez et al, 2007). Other focal groups for higher-level taxonomic studies in my lab include Esociformes (pikes and relatives, López et al., 2004), Clupeiformes (sardines, herrings and relatives, Li and Ortí 2007), Gadiformes (cod, hakes and relatives, Roa-Varón and Ortí, in prep), Perciformes (Mahon et al., in prep), and the “lower” actinopterygian lineages (Ortí and Li, in prep). At shallower taxonomic levels, previous work examined relationships among genera in the piranha subfamily Serrasalminae, Characiformes (Ortí et al. 1996). This project continued in my lab at UNL as part of a M.S. thesis by Arjun Sivasundar (Ortí et al. 2007) and through collaborations with W. Dahdul (PhD student with J. Lundberg). Other examples of lower-level phylogenies include studies on catostomids (blue suckers) by M. Bessert (Bessert, Sitzman and Ortí, 2007; Bessert and Ortí 2007; and in prep) and on gerreid mojarras (Chen et al., 2007).
More recently, bioinformatic approaches using available genomic data have focused on phylogenetic relationship among fish model organisms (Chen et al., 2004), the evolution of gonadotropin releasing hormones (GnRH) in fishes (Guilgur et al., 2007) and the development of new molecular markers for fish phylogenetics (Li et al., 2007 – BMC article). Development of a phylogenomic approach for fish systematics is now possible in part due to the results presented in this latter paper. More than 100 carefully selected, single-copy, protein-coding genes with long exons (> 800 bp) have been identified and will provide the molecular basis for assembling the tree of life of fishes. Preliminary analyses of some of these markers across diverse taxonomic groups are encouraging (Li, Lu, and Orti, submitted).
Clearly, the sheer size of the problem dictates that no individual effort could possibly lead to the ultimate solution, but my lab has contributed significantly to advance this field of study. One of the main results obtained by the CAREER project was the establishment of the nuclear gene RAG-1 as the first widely used molecular marker for fish phylogenetics. Several studies using this gene for phylogenetics (including our own) were published for many groups of fishes. Recent work (Li et al., 2007, BMC paper) has made available the next set of phylogenomic markers that will be used by the Tree of Life Project. A database with PCR primers and molecular protocols for the most useful and popular genetic markers for fish phylogenetics is under construction at the DeepFin web site, to facilitate the common use of markers by other research groups. My main achievement as the PI for the DeepFin research network, however, has been to foster and coordinate the formation of a multi-institutional team that could generate long term funding for this project. Tree of Life funding from NSF finally came in 2007 for our team of collaborators from 8 institutions across the USA (Team EToL).
Current and future work: Research coordination activities continue to have a central role now that we have established a diverse but functional research group disseminated across North America. Plans are required to further develop networking tools in our interactive portal as well as a bioinformatic infrastructure capable of handling huge volumes of data with user-friendly interfaces. The products of the Tree of Life effort need to be widely disseminated to the general public as well as to the scientific community. Partnerships between DeepFin and the Tree of Life Web and the Encyclopedia of Life will be critical to achieve this goal.
Experimental work to assemble the new genetic database for phylogenetic analyses is just beginning. Ten new candidate molecular markers have been optimized and at least 10 more will be optimized in my lab. Chenhong Li defended his PhD thesis in August 2007 and is now a postdoctoral associate in charge of continuing this aspect of the research. Tissues from exemplar taxa for this study are being distributed to all labs involved in collecting the new molecular evidence. This is an exciting time that will provide new opportunities for graduate and undergraduate student training in my lab for many years to come.
Funding has been obtained from the NSF (PIRE Program, OISE-0530267, from 2005 to 2010) and from the Research Agency of Argentina (RAICES Program) for the Patagonia project. The Amazonian project has preliminary funding through the generous support from the UNL Office of Research (Cluster Grant for Amazonian fishes, from 2007 to 2008) and through a Doctoral Fellowship from NSF to my PhD student Stuart Willis.
Rationale:At the opposite end of the phylogenetic spectrum, this research falls within the realm of population genetics, an area of interest that developed during my postdoctoral experience with John Avise. At this level, I am interested in integrating data on gene genealogies obtained from conspecific individuals with evidence of historical demography and geographic distribution (phylogeography). Using intraspecific gene genealogies “mapped” onto geographic space the goal is to gain historical perspectives on genetic population structure. As populations are isolated over time and space, incipient species may originate. Phylogeographic analyses, therefore, lie at the interface between population genetics and phylogeny. When phylogeographic structures of several co-distributed taxa are compared, the impact of historical events on evolutionary diversification of regional biotas can be tested. Very little is known about the processes shaping the origin of diversity in Patagonia and Amazonia, two unique regions of the world.
Background and progress to date: My first contribution in this area was a global survey of mtDNA sequence variation in threespine sticklebacks that suggested a recent colonization of the Atlantic basin by fish from the eastern Pacific (Ortí et al., 1994). Several years later, we published a study on the systematics and phylogeography of a group of Neotropical freshwater fishes (Prochilodus, Characiformes), widely distributed in all major river basins of South America, including the Amazon (Sivasundar et al., 2001). Prochilodus fishes have been a long-standing interest for me, since I was an undergraduate student in Argentina. Work on Amazonian fishes continues, with extensive sampling efforts by my Brazilian colleagues (Dr I. Farias and her students at the University of Amazonas in Manaus) and graduate student S. Willis. As a result, our tissue collections now include several species and a large geographic area. A new molecular marker (intron 3 of the nuclear gene ependymin) has been optimized for Prochilodus, showing very high levels of intra- and interspecific variation, suggesting that it would be a marker of choice for other taxa. Microsatellite assays have been developed to increase the number of available markers already available for these fishes. Dr. Carmen Rosa Garcia Dávila, a colleague biologist from the Instituto de Investigaciones de la Amazonía Peruana (IIAP) from Peru, was a visiting scholar in my lab during the fall of 2005. During her visit, she was trained in mirosatellite typing techniques to assess differentiation among populations of Prochilodus in separate headwater tributaries of the Amazon in Peru (work in progress). A second focus of the Amazonian fish project is the doctoral thesis project of S. Willis that involves several species of cichlid fishes. Stuart has traveled to the Amazon several times to collaborate with Dr. Farias. A manuscript analyzing the effect of current connections between the Orinoco and Amazonas basins on the distribution of several species of Cichla (Cichlidae) is in preparation (Willis et al., in prep).
A recent development of our Amazonian fish project has been the formation of a research cluster at UNL—originating from the Program of Excellence in Population Biology— that combines expertise in ecology, evolution, mathematics, geology, and education to develop an integrated approach. Our group has come up with new ideas to model the joint effects of ecology, history, and chance in explaining biodiversity, focusing on fish species adapted to the rapids (cachoeiras) in the vast Amazonian basin. A preproposal to the NSF-PIRE program was submitted by the group last year but, unfortunately, it was not recommended for funding. The generous support from the office of Research at UNL is now supporting the continuity of this effort for one year to help us produce a more competitive proposal. The figure summarizes the main components of this project.
Our focus on Patagonian fishes is more recent. Through collaboration with scientists from Brigham Young University, Dalhousie University, and counterparts in Patagonia (Argentina and Chile), my lab is part of an international effort to study comparative phylogeography of native species from Patagonia, including fishes, lizards, frogs, freshwater crabs, and plants. This effort is funded by the NSF’s PIRE Program. My lab is involved with the study of silversides (freshwater and marine fishes in the family Atherinopsidae), one of the native fish species included in the study. A PhD student in my lab (Julie Sommer) is developing her thesis in this area. Most of the field-work necessary to collect specimens for our study has been completed and molecular markers (nuclear and mtDNA loci and microsatellites) are being optimized at this time. Preliminary results have been presented at national meetings this year (ASIH meetings, St. Louis, MO, July 2007). Interactions with graduate students from Argentina and Chile have been established and plans have been made to facilitate exchange programs among participating labs.
Current and future work: The Patagonian silversides project is in its second year and progress is good. In addition to the phylogeography focus for the freshwater species originally included in the NSF proposal, the initial scope of the project has been expanded to produce a comprehensive phylogenetic study of the tribe (Sorgentinini) that includes all the marine and freshwater silversides of South America. Collaborations with other scientists from Chile, Argentina, and Brazil have been established to secure appropriate taxonomic sampling outside of Patagonia. My PhD student J. Sommer is working on a proposal to be submitted to NSF’s DDIG program (Dissertation improvement grant) in November 2007. Two doctoral students from Argentina (Mariela Cuello from Universidad de La Plata and Cecilia Contegrand from Universidad del Comahue) are scheduled to visit UNL to work on molecular techniques and to analyze silverside specimens during the spring of 2008. Their visit and their doctoral theses are being funded in part by a grant recently awarded to our team by the Argentinean Research Agency (RAICES grant, in collaboration with Drs. V. Cussac from Comahue in Argentina and D. Ruzzante from Dalhousie, Canada).
Current work for the Amazonian fishes project includes the following: (1) collecting permits for the Amazonian project have been solicited through the efforts of Dr Farias to collect endemic species from the rapids (cachoeiras) in several localities of the Amazonian basin. A collecting trip is in the planning stages. (2) I recently established a collaboration with Dr Pedro Galetti, from the Universidade Federal de São Carlos (Sao Paulo State, Brazil) facilitating a student exchange program for one of his doctoral students (Mr. Luis Costa), who will visit my lab during 2008 (with funding from Brazilian sources). He will use this visit to collect data for his doctoral thesis on the migratory freshwater catfish Pseudoplatystoma. (3) Finally, another collaboration was established with Dr. Luz F. Jimenez Segura from the Universidad de Antioquia (Colombia), who is studying migratory fishes in the Magdalena river. We have established a formal framework agreement of cooperation between UNL and Antioquia to facilitate student exchanges and were successful to obtain funding (from Colombian sources) to bring her students to UNL next spring to start the collection of molecular data.
Among several other projects, three additional lines of research in my lab that I would like to highlight in this narrative were product of students’ projects or collaboration with Dr C. Wood from the Nebraska Center for Virology. All these projects were supported by extramural funding.
"Evolution of Clade C HIV-1 in Infected Children"
A significant new research direction developed in 2000 through interactions with the Nebraska Center for Virology, resulting in NIH funding (R01 grant for $1,586,250, 2001–2006) to study perinatal transmission and evolution in infected mother–infant pairs (MIPs) in Zambia (Zhang et al., 2002, 2005, 2006). A postdoctoral associate (Dr Federico Hoffman) conducted part of this research in my lab.
The causal mechanisms of differential disease progression in HIV-1 infected children remain poorly defined, and much of the accumulated knowledge comes from studies of subtype B infected individuals. In our studies, we longitudinally characterized the evolution of the Env V1–V5 region from subtype C HIV-1 perinatally infected children with different clinical outcomes. We investigated the possible influence of viral genotype and humoral immune response on disease progression in infants. Phylogenetic analyses clearly indicated two contrasting phylogenetic transmission patterns, with some cases suggesting selective transmission of a single or a few variants, and others transmitting multiple maternal HIV-1 variants to the infant. In one case of a slow progressor infant with delayed onset of AIDS, we demonstrated a correlation between HIV-1 Env evolution and the humoral immune response. In this case, there was an increase in genetic diversification in the infant viral sequences after 12 months, which coincided with increases in neutralizing antibody titers. In addition, we documented episodes of viral growth and successive immune reactions extending 5–6 years after birth. Our data suggest that neither genetic variation in Env, or initial maternal neutralizing activity, or the level of passively acquired neutralizing antibody, or the level of the de novo neutralization response appear to be linked to differences in disease progression in subtype C HIV-1 infected children.
“Paternity, cooperative breeding and genetic chimerism in marmosets”
My second PhD student, Cory Ross, was a behavioral physiologist (a primatologist), who had worked with Dr J. French (from UN-Omaha) during her MS thesis. For her doctoral research, we secured an NSF grant (IBN-0417202 for $161,198, 2004-006) for her study on genetic chimerism in New World monkeys (marmosets).
The formation of viable genetic chimeras through the transfer of cells between siblings in utero is rare in mammals, but not in marmostes. The main finding from this study (Ross et al., 2007) was that genetic chimerism in marmosets (Callithrix kuhlii) is not limited to blood-derived hematopoietic tissues as was previously described. All somatic tissue types sampled in our study were found to be chimeric. Notably, chimerism was demonstrated to be present in germ-line tissues, an event never before documented as naturally occurring in a primate. In fact, we found that chimeric marmosets often transmit sibling alleles acquired in utero to their own offspring. Thus, an individual that contributes gametes to an offspring is not necessarily the genetic parent of that offspring. The presence of somatic and germ-line chimerism may have influenced the evolution of the extensive paternal and alloparental care system of this species. Although the exact mechanisms of sociobiological change associated with chimerism have not been fully explored, we showed that chimerism alters relatedness between twins and may alter the perceived relatedness between family members, thus influencing the allocation of parental care. Consistent with this prediction, we found a significant correlation between paternal care effort and the presence of epithelial chimerism, with males carrying chimeric infants more often than nonchimeric infants. Therefore, we propose that the presence of placental chorionic fusion and the exchange of cell lines between embryos may represent a unique adaptation affecting the evolution of cooperative care in this group of primates.
“Conservation Genetics of the Blue Sucker fish (Cycleptus elongatus, Catostomidae)”
This was the doctoral research of my fourth PhD student, Mike Bessert, funded by the Nebraska Game and Parks Comission (State Wildlife Grant to M. Bessert for 22,000, 2003-2007). Major findings have been published or are in preparation.
The overarching theme of this research was to investigate hierarchical levels of relatedness in natural populations of the cycleptid fishes (blue suckers), a widespread genus in North America. In order to do so, Mike acquired one of the most expansive (tissue) collections for any North American freshwater fish taxon – both numerically and in terms of geographic coverage- by establishing a nation-wide network of collaborators among workers of several state agencies. Novel genus-specific molecular markers were isolated from genomic DNA and optimized for these fishes, effectively addressing the fact that they are tertaploids (Bessert, Sitzman, and Ortí 2007). These loci enabled the use of sophisticated techniques to estimate phylogeny, phylogeography, and population genetic structure (Bessert and Ortí, in prep). In addition, they were used to assess population trends along a major section of the Missouri River that has been dramatically modified in the past 70 years (Bessert and Ortí, 2007). The results of this work provide important contributions to a range of biological sub-disciplines, including: (1) systematics (phylogenetic delineation of a new species), (2) molecular techniques (development of a new technique for isolating paralogous microsatellite loci in polyploid organisms), and (3) conservation genetics (assessment of historical and contemporary demography and population structure). For the cycleptid fishes, the latter is of greatest urgency given the conservation status of the group.